There is a continuing need for an inexpensive, easy to perform method of detecting substances that are present in fluids at concentrations on the order of 1.times.10.sup.-6 grams or less. Prior art methods capable of accurately detecting a substance in a fluid at these concentrations are cumbersome, expensive and require long periods of time to perform. In addition, expensive and complicated equipment is required to perform these prior art methods.
There are many prior art assay methods designed to detect the presence of soluble substances in serum and other media of biological importance. For substances that have biological activity, one can simply measure the activity in the biological fluid to detect the presence of the substance. For example, one can measure the presence of the enzyme acid phosphatase in blood serum by adding a substrate of acid phosphatase enzyme such a p-nitrophenyl phosphate and incubating the solution for a period of time. If the enzyme is present in the blood, the solution will turn yellow as the substrate is hydrolyzed by the enzyme to phosphate and p-nitrophenol. However, there are many problems associated with this type of assay. For example, the substance to be assayed must have a biological activity that can be measured. Often the measurement of biological activity can be cumbersome and very time consuming. Furthermore, the activity of the enzyme may be inhibited by the presence of an inhibitor. If such an inhibitor is present, a falsely low activity will be measured. In addition the enzyme may be present in the fluid but may be inactive.
Another method of measuring the presence of trace substances in biological fluids is a process known as chromatography. There are many different types of chromatography. Thin layer chromatography, in combination with mass spectroscopy or gas phase chromatography, has been used to isolate and quantify a particular substance in biological fluids. However, thin layer chromatography has a number of deficiencies such as being slow, being subject to a wide range of interfering materials, and suffering from severe fluctuations inreliability.
Liquid chromatography is another method of isolating materials from biological fluids. In this method, advantage is taken of particular molecule's physical properties, such as size or charge. However, one still must utilize a method of analyzing the particular substance after it is isolated. This can be done by measuring biological activity, absorbance characteristics, mass spectroscopy, or by further separation analysis.
Another method that takes advantage of molecular charge and size is gel electrophoresis. In this method, a biological sample is placed on a porous gel. The sample and the gel are then subjected to an electrical field causing the ample to migrate through the gel. The rate of migration is dependent upon the charge and on the size of the molecule. In this way, different molecules can be separated and isolated.
There are many problems with chromatographic and electrophoretic methods for identification of substances. One of the problems is in identifying the substance after it has been isolated. In order to identify the isolated substance, one must perform another procedure such as measurement of biological activity, analysis by mass spectroscopy or identification by other methods, such as immunological methods. An electrophoresis or chromatography procedure is a time consuming process taking several hours to several days. In addition, the equipment used in these procedures is expensive and requires an experienced technician to perform the analysis.
Another method of identifying trace amounts of a particular substance in a solution is through immunological techniques. All immunological procedures use an antigen, and an antibody which is specific for the antigen. Prior art immunological methods include immunological precipitation in which the antibody combines with an antigen for which the antibody is specific. The resulting complex precipitates out of solution forming a visible precipitate.
Agglutination is another prior art method of detecting small concentrations of a particular substance. In agglutination, a body, such as a red blood cell or a bacteria, is reacted with antibodies that are specific for an antigen on the surface of the body. As the antibodies react with the surface antigens, the cells agglutinate forming a dense, visible clump.
The procedures of immunoprecipitation and immunoagglutination suffer from a general lack of sensitivity. In addition, the procedures require the antigen to have multiple antibody binding sites so that the antibodies may crosslink the antigens causing the precipitation or agglutination. The process of immunoprecipitation requires several hours to several days to complete thereby making the procedure impractical for many situations where the identification or quantification of a particular substance must be performed quickly.
The problem of lack of precision by the above described procedures was overcome by the procedure known as radioimmuno assay. In this procedure, the antigen to be measured is "labeled" with a radioactive element to form a radioactive analogue. Radioactive isotopes that are commonly used in radioimmunoassays are shown in Table I.
TABLE I ______________________________________ Radioactive Isotopes used for Tagging Biological Materials Specific Activity of Pure Isotope Isotope (Curies per mole) Half-life ______________________________________ C.sup.14 6.25 .times. 10.sup.1 5230 years H.sup.3 2.91 .times. 10.sup.4 12.3 years S.sup.35 1.50 .times. 10.sup.6 87 days I.sup.125 2.18 .times. 10.sup.6 60 days P.sup.32 3.16 .times. 10.sup.6 14.3 days I.sup.131 1.62 .times. 10.sup.7 8.1 days ______________________________________
By mixing an antibody with solutions of a hapten or antigen to be analyzed, and with the radioactive antigen analogue, the radioactive analogue will be prevented from binding to the antibody to an extent directly proportional to the concentration of the hapten or antigen in the solution. By then separating and assaying the free radioactive analogue from the antibody bound radioactive analogue, one can indirectly determine the amount of hapten or antigen in the original-solution.
However, the use of radioisotopes in such an assay is a potential health hazard and, furthermore, the instrumentation required for radioimmunoassay is relatively sophisticated and expensive. Another problem with the radioimmunoassay is in labeling the antigen or antibody. The isotopes that are most commonly used are those with a short half-life. These include Iodine-131 and Iodine-125. Because these isotopes have such a short half-life (8.1 days and 60 days, respectively), the labeled component of an assay must periodically be replaced with new product. In addition, a standard curve must be prepared with each unknown sample since the specific activity of the isotope is constantly decreasing. A further problem with some labeled components is autodegradation. The isotopes that are commonly used to label the compounds are relatively strong radiation emittors and can cause the compounds to which they are attached to be degraded Finally, with the advent of increasing number of government regulations concerning the disposal of radioactive wastes, disposing of the radioactive isotopes used in radioimmuno assays has become an increasingly difficult and expensive problem.
Enzyme immunoassays overcome the above problems and in addition, have the unique advantage of potential amplification of the measured activity. (The field of enzyme immunoassays has been extensively reviewed in Developments in Immunology, Vol. 18, Immunoenzymatic Techniques, Elsevier Science Publishers, 1983) This method replaces the radioactive biological substance analogue with an enzyme labeled biological substance (hapten or antigen). Typical enzymes that can be used as labels in the enzyme immunoassay are listed in Table II.
TABLE II ______________________________________ Enzymes commonly used in enzyme immuno assay ______________________________________ Alkaline phosphatases Glucose oxidases Ureases Peroxidases .beta.-Galactosidases Glucose-6-phosphate dehydrogenases Lysozymes Malate dehydrogenases ______________________________________
Such modified enzyme molecules retain their enzymatic activity and the enzyme-labeled biological substance will compete for antibody complex formation with the unknown amount of free biological substance in the system. The complexes may be separated in view of the insolubility in certain substances. The activity of the separated complex, or the part remaining in solution, is used as a measure of the amount of antigen originally present. The same principle may be applicable to a reverse system, using enzyme-labeled antibodies whenever the unmodified version of the same antibody present in biological fluids has to be determined.
There are several variations of the enzyme immuno assay. In one variation, known as enzyme-linked immunosorbent assay (ELISA), labeled and unlabeled antigen compete for attachment to a limited quantity of solid-phase antibody. The enzyme label that is displaced is measured, and the calculations that follow are essentially the same as in radioimmunoassay procedures.
The sandwich technique is another variation of enzyme immunoassay and relies on the multivalence of antigens and capacity to bind simultaneously with two molecules of antibody. The first antibody molecule is a solid phase reactant. It is used in excess to ensure binding of all the antigen molecules in the unknown sample. After that reaction is completed, an enzyme-labeled antibody is added and incubated with the complex resulting from the first phase. The labeled antibody then combines with available determinants on the antigen. Excess antibody is removed by washing and enzyme activity is then determined. As in other systems, the amount of enzyme bound to the complex is an indirect measure of the amount of antigen in the sample. Variations of this method include the second antibody method. In that method, antigen is reacted first with solid phase antibody and later with free antibody, neither of which is labeled. Then enzyme-labeled antibody with a specificity for the free antibody is used as the last reagent.
Most of the enzyme immunoassay techniques are classified as heterogeneous assays. This means that the bound labeled molecule must, at some point in the assay procedure, be separated from the free labeled molecule in order to perform the necessary calculations to determine the amount of unknown substance in the fluid. This requires a separation step in the assay and adds to both the time and expense of the assay procedure. There are enzyme immuno assay procedures that are homogeneous assays in that there is no separation of bound labeled substance and unbound labeled substance. Such a system does not require a solid phase reactant, but rather relies on an inhibition of enzyme activity by the combination of an antibody with an enzyme-labeled antigen or hapten. This type of assay is of limited usefulness since not all antigen-antibody combinations will result in a predictable diminution of enzyme activity.
Enzyme immunoassays are generally as sensitive as radioimmunoassays and are much safer because no radioactive isotopes are used. In addition, enzyme immunoassays generally require less sophisticated equipment than the radioimmunoassays. An enzyme immunoassay is generally much less expensive than a corresponding assay done by radioimmunoassay.
However, there are still significant problems associated with the typical enzyme immunoassay. The time required to run an enzyme immunoassay, for many applications, is too long. In most cases, an incubation period of at least several hours is required to perform the assay. In addition, the typical enzyme immunoassay comprises several washing steps and an additional incubation step with an enzyme substrate to develop a color which can be measured. The color from the enzyme reaction must then be measured in a spectrophotometer.